Early reports of MB’s impact on tau suggest that the compound is capable of directly inhibiting the growth of tau polymers.17,18
Recent data show that MB is capable of modulating tau levels through additional mechanisms such as inhibition of Hsp70 ATPase activity24
and our findings suggest that another mechanism by which MB reduces tau levels is through the induction of autophagy as we have observed altered markers of autophagic clearance in cells, primary neurons, organotypic slice cultures and mice treated with nanomolar levels of MB.
Our studies have shown that chronic organotypic slice cultures prepared from tau transgenic mice, incubated with nanomolar concentrations of MB have decreased levels of hyperphosphorylated soluble tau and insoluble, aggregated tau. Concurrently, decreased levels of p62 and increased levels of catD, BECN1 and LC3-II were seen relative to vehicle-treated hemi-brains from the same mice. Similar results were seen in vivo as oral treatment of tau transgenic mice with MB at multiple doses over the course of two weeks led to a significant reduction in hyperphosphorylated soluble tau levels, and significantly reduced levels of p62, with increased levels of catD, BECN1 and LC3-II. The results in vivo did not fully replicate the organotypic slice data as insoluble tau levels were not significantly changed in the live mice for reasons possibly related to insufficient brain concentrations of MB, or inter-mouse variability. In addition, there was a difference in the effect on total tau levels (unchanged in slices, decreased in vivo) and some of the phospho-tau epitopes (such as PHF-1) that could reflect the model systems or exposure to drug. However, the induction of autophagy was consistent in all systems tested, including CHO cells and primary neurons.
One of the key features of autophagy is the formation of double-membrane bound vesicles, called autophagosomes or autophagic vesicles (AVs) that fuse with lysosomes during flux leading to degradation of the contents. Induction of AV formation and flux is a complex series of events that can be triggered either through suppression of mTOR activity, or independent of mTOR. mTOR activation (autophagy suppression) can occur via IGF signaling in a pathway that includes Akt and PI3 kinase CI. Typically, increased phosphorylation and hence activity of Akt in turn leads to increased phosphorylation and activity of mTOR, which in turn suppresses autophagy. However, transgenic animals treated with MB showed a marked increase in phosphorylated Akt but a decrease in one of the phosphorylation sites of mTOR (ser2448) that is an (indirect) Akt target.34
Although the events occurring between Akt and mTOR are not clear, similar results have been shown with rapamycin. Rapamycin treatment increases PI3 kinase C1 activity which increases Akt activity (with some groups reporting increased phosphorylation at ser308, others at both ser308 and ser47335
), which inhibits GSK-3 activity though phosphorylation of the inhibitory site at ser9,36
but increased Akt activity following rapamycin exposure suppresses the activity of mTOR and thus induces autophagy. It was found that this effect was mediated through the insulin receptor substrate (IRS).36
IRS is a target of mTOR and thus reduction of mTOR activity prevents IRS degradation. Preventing IRS degradation results in increased IGF receptor signaling and thus greater phosphorylation of Akt and GSK-3β ser9. A mechanism similar to rapamycin by which MB could stimulate autophagy is presented in . In cells, both rapamycin and MB reduced mTOR phosphorylated at ser2448 and reduced phosphorylation of the mTOR target p70 at thr389 indicating reduced activity of the kinase. Further downstream effects on IRS and Akt phosphorylation, and conversion of LC3 were also observed with both drugs. However, it appears that the two drugs have a somewhat different timecourse with regards to effects on tau, with rapamycin requiring longer incubation for significant tau clearance (data not shown). In addition to mTOR mediated effects on autophagy, effects mediated by GSK-3 may also have played a role as inhibition of GSK-3 activity due to increased activity of Akt has been shown to stimulate AV nucleation by promoting interaction between Bax-inducing factor 1 (SH3GLB1/Bif-1) and BECN1 Atg637,38
in a large protein complex.32,38
Furthermore, because GSK-3 is one of the major kinases involved in tau phosphorylation, decreased GSK-3 activity may have led directly to lower levels of phospho-tau.
Figure 10. Proposed mechanism by which MB could stimulate autophagy through mTOR and Akt. (1) Inhibition of mTOR induces autophagy directly but also reduces phosphorylation of IRS1. (2) Dephosphorylation of IRS-1 results in decreased degradation (more ...)
Two other studies have examined the effect of small molecules based on the phenothiazine scaffold, similar to MB, on autophagy induction.39,40
These studies also demonstrated autophagy induction, however, they found no change in the phosphorylation status of either mTOR or p70, suggesting an mTOR-independent mechanism. Our data show that detecting changes in phosphorylation of target proteins such as p70 is highly time dependent. For example, in CHO cells, reduction in pp70 was seen at 6 h, but not at three or 18 h (data not shown). Detection can also be affected by model system used. In transgenic animals, MB produced a significant reduction in mTOR phosphorylated at ser2448, but changes in pp70 status were not detected. In addition to differences in the experimental design, the small molecules utilized in these studies contain large meso substituents not present in MB that may affect their function.
An additional mechanism by which MB could potentially affect autophagy is through interaction with heat shock proteins. Jinwal et al. (2009)24
and Wang et al. (2010)21
demonstrated that MB is an inhibitor of Hsp70 ATPase activity. Reduction in Hsp70 activity resulted in a decrease in the levels of total and phosphorylated tau. The authors hypothesized that Hsp70 plays a protective role, preventing the degradation of the tau protein. Interfering with the tau-Hsp70 interaction could therefore allow tau to be cleared. Further, Hsp70 binding proteins have been shown to alter LC3-II levels in cells, indicating that modulation of Hsp activity has an effect on protein degradation systems. In addition, Hsp and Hsc70 are involved in chaperone-mediated autophagy (CMA), an alternative protein degradation pathway, and inhibition of CMA can increase autophagy.41
Jinwal et al. (2009)24
reported that MB inhibits Hsp70 activity with an EC50
of ~80 μM but the lowest dose used in this study was 5–10 fold below the lowest dose used in Wang et al. (2010).21
Even given the accumulation of MB in brain tissue, it is unlikely that the lowest doses of MB used in our study were sufficient to alter the ATPase activity of Hsp70. However, in the transgenic animals given the highest dose (20 mg/kg) it is possible that several mechanisms were altered.
In contrast to our results, one study demonstrated that MB administered to mice over a similar dose range had no effect on tau levels or autophagy induction markers such as LC3-II.21
In this case, the route of administration differed from ours—MB was added to food powder rather than by oral gavage which may have had a significant effect on bioavailability and levels in the brain which could account for the difference in outcomes. In general, these contradictory findings highlight the importance of dosing and methodology when evaluating drug efficacy.
As the tau protein is abnormal in several major neurodegenerative diseases, it represents an attractive target for disease-modifying therapy. Methylene blue has been shown to reduce tau, improve cognition and attenuate neurodegeneration in a tau transgenic line25
suggesting that in addition to its effects on the levels of disease-associated proteins, it can impact important functional outcomes. We have also observed an improvement in cognitive performance after administration of MB, in the same tau transgenic mouse line published25
(see Supplementary Data
). MB has also been shown to be neuroprotective in other animal models of degeneration. Amounts as low as 70 μg/kg have been shown to reduce lesion size and neurotoxicity in a rat model of stroke28,42
and neuroprotective effects were also seen in an animal encephalopathy model.43
Our data provide further in vivo support for the use of MB to protect against disease, and suggest that autophagy modulation is a valid avenue for drug development for tauopathies.